Adenosine monophosphate-activated protein kinase (AMPK) is a conserved sensor of intracellular energy activated in response to low nutrient availability and environmental stress. In a screen for conserved substrates of AMPK, we identified ULK1 and ULK2, mammalian orthologs of the yeast protein kinase Atg1, which is required for autophagy. Genetic analysis of AMPK or ULK1 in mammalian liver and C. elegans revealed a requirement for these kinases in autophagy. In mammals, loss of AMPK or ULK1 resulted in aberrant accumulation of the autophagy adaptor p62 and defective mitophagy. Reconstitution of ULK1-deficient cells with a mutant ULK1 that cannot be phosphorylated by AMPK revealed that such phosphorylation is required for mitochondrial homeostasis and cell survival following starvation. These findings uncover a conserved biochemical mechanism coupling nutrient status with autophagy and cell survival.

SUMMARY Autophagy, the primary recycling pathway of cells, plays a critical role in mitochondrial quality control under normal growth conditions and in the response to cellular stress. The Hsp90-Cdc37 chaperone complex coordinately regulates the activity of select kinases to orchestrate many facets of the stress response. Although both maintain mitochondrial integrity, the relationship between Hsp90-Cdc37 and autophagy has not been well characterized. Ulk1, one of the mammalian homologues of yeast Atg1, is a serine-threonine kinase required for mitophagy. Here we show that the interaction between Ulk1 and Hsp90-Cdc37 stabilizes and activates Ulk1, which in turn is required for the phosphorylation and release of Atg13 from Ulk1, and for the recruitment of Atg13 to damaged mitochondria. Hsp90-Cdc37, Ulk1 and Atg13 phosphorylation are all required for efficient mitochondrial clearance. These findings establish a direct pathway that integrates Ulk1- and Atg13- directed mitophagy with the stress response coordinated by Hsp90 and Cdc37.

Mutations in VCP cause multisystem degeneration impacting the nervous system, muscle, and/or bone. Patients may present with ALS, Parkinsonism, frontotemporal dementia, myopathy, Paget’s disease or a combination of these. The disease mechanism is unknown. We developed a Drosophila model of VCP mutation-dependent degeneration. The phenotype is reminiscent of PINK1 and parkin mutants, including a pronounced mitochondrial defect. Indeed, VCP interacts genetically with the PINK1/parkin pathway in vivo. Paradoxically, VCP complements PINK1 deficiency but not parkin deficiency. The basis of this paradox is resolved by mechanistic studies in vitro showing that VCP recruitment to damaged mitochondria requires Parkin-mediated ubiquitination of mitochondrial targets. VCP recruitment coincides temporally with mitochondrial fission, and VCP is required for proteasome-dependent degradation of Mitofusins in vitro and in vivo. Further, VCP and its adaptor Npl4/Ufd1 are required for clearance of damaged mitochondria via the PINK1/Parkin pathway, and this is impaired by pathogenic mutations in VCP.

In the original Supplemental Information for this Article, the same image was used for both Figure S4 and Figure S5. This has been corrected in the Supplemental Information online.

The ubiquitous m-and -calpains are thought to be localized in the cytosolic compartment, as is their endogenous inhibitor calpastatin. Previously, -calpain was found to be enriched in mitochondrial fractions isolated from rat cerebral cortex and SH-SY5Y neuroblastoma cells, but the submitochondrial localization of -calpain was not determined. In the present study, submitochondrial fractionation and digitonin permeabilization studies indicated that both calpain 1 and calpain small subunit 1, which together form -calpain, are present in the mitochondrial intermembrane space. The N terminus of calpain 1 contains an amphipathic ␣-helical domain, and is distinct from the N terminus of calpain 2. Calpain 1, but not calpain 2, was imported into mitochondria. Removal of the N-terminal 22 amino acids of calpain 1 blocked the mitochondrial calpain import, while addition of this N-terminal region to calpain 2 or green fluorescent protein enabled mitochondrial import. The N terminus of calpain 1 was not processed following mitochondrial import, but was removed by autolysis following calpain activation. Calpain small subunit 1 was not directly imported into mitochondria, but was imported in the presence of calpain 1. The presence of a mitochondrial targeting sequence in the N-terminal region of calpain 1 is consistent with the localization of -calpain to the mitochondrial intermembrane space and provides new insight into the possible functions of this cysteine protease.Calpains (EC 3.4.22.17) are a family of Ca 2ϩ -activated cysteine proteases, including both ubiquitous and tissue-specific isoforms, that cleave their substrate proteins at discrete sites to modulate activity (1-3). The best characterized, and the predominant calpains in the central nervous system, are the classical m-and -calpains. Their physiological roles have not been fully elucidated but include cell motility, cell differentiation, membrane fusion, platelet activation, and signal transduction (3). Also extensively investigated have been the pathological roles of calpains in cell death, where calpains can cleave key structural proteins and contribute to the release of death-related proteins such as apoptosis-inducing factor (AIF) 3 (4 -9). At present, it is unclear whether the -and m-calpains have distinct or overlapping functions. They are each heterodimers consisting of a unique 80-kDa large catalytic subunit (calpain 1 or 2) and a common 28-kDa small regulatory subunit (calpain small subunit 1 or 2) (2). In vitro, the substrates of m-and -calpains are similar, if not identical (10). Knock-out of the -calpain large subunit, calpain 1, results in viable mice with reduced platelet aggregation and impaired tyrosine phosphorylation in platelets, but not overt phenotype (11). Knock-out of the m-calpain large subunit, calpain 2, or of calpain small subunit 1 (CSS1) is embryonically lethal (12)(13)(14).Both m-and -calpains are considered to be cytosolic enzymes (2,3, 15,16). An association of m-and -calpains with subcellular organelles including endoplasmic ret...

Reactive oxygen species (ROS) may cause cellular damage and oxidative stress-induced cell death. Autophagy, an evolutionarily conserved intracellular catabolic process, is executed by autophagy (ATG) proteins, including the autophagy initiation kinase Unc-51-like kinase (ULK1)/ATG1. Although autophagy has been implicated to have both cytoprotective and cytotoxic roles in the response to ROS, the role of individual ATG proteins, including ULK1, remains poorly characterized. In this study, we demonstrate that ULK1 sensitizes cells to necrotic cell death induced by hydrogen peroxide (H 2 O 2 ). Moreover, we demonstrate that ULK1 localizes to the nucleus and regulates the activity of the DNA damage repair protein poly (ADP-ribose) polymerase 1 (PARP1) in a kinase-dependent manner. By enhancing PARP1 activity, ULK1 contributes to ATP depletion and death of H 2 O 2 -treated cells. Our study provides the first evidence of an autophagy-independent prodeath role for nuclear ULK1 in response to ROS-induced damage. On the basis of our data, we propose that the subcellular distribution of ULK1 has an important role in deciding whether a cell lives or dies on exposure to adverse environmental or intracellular conditions. Cell Death and Differentiation (2016) 23, 216-230; doi:10.1038/cdd.2015; published online 3 July 2015Reactive oxygen species (ROS), such as superoxide and hydrogen peroxide (H 2 O 2 ), are formed by the incomplete reduction of oxygen during oxidative phosphorylation and other enzymatic processes. ROS are signaling molecules that regulate cell proliferation, differentiation, and survival. 1-3 Accumulation of ROS (i.e., oxidative stress) on exposure to xenobiotic agents or environmental toxins can cause cellular damage and death via apoptotic or nonapoptotic pathways. [4][5][6] Oxidative stress-induced cellular damage and death have been implicated in aging, ischemia-reperfusion injury, inflammation, and the pathogenesis of diseases (e.g., neurodegeneration and cancer). 7 Oxidative stress also contributes to the antitumor effects of many chemotherapeutic drugs, including camptothecin 8,9 and selenite. 10,11 Autophagy, an evolutionarily conserved intracellular catabolic process, involves lysosome-dependent degradation of superfluous and damaged cytosolic organelles and proteins. 12 It is typically upregulated under conditions of perceived stress and in response to cellular damage. The consequence of autophagy activation -whether cytoprotective or cytotoxicappears to depend on the cell type and the nature and extent of stress. Although most studies indicate a cytoprotective role for autophagy, some evidence suggests that it contributes to cell death in response to oxidative stress. [13][14][15][16][17] Studies have also indicated that autophagy may be suppressed in response to oxidative stress, thereby sensitizing certain cells to apoptosis. 18,19 Unc-51-like kinase/autophagy 1 (ULK1/ATG1) is a mammalian serine-threonine kinase that regulates flux through the autophagy pathway by activating the VPS34 PI(3) kinas...

No abstract

• Mitochondrial dysfunction in aged mtDNA-mutator mice is associated with activation of mechanistic target of rapamycin and suppression of autophagy in erythroid cells.• Autophagy maintains mitochondrial function in erythroid progenitors of mtDNA-mutator mice, and disrupting it accelerates onset of anemia.Somatic mitochondrial DNA (mtDNA) mutations contribute to the pathogenesis of age-related disorders, including myelodysplastic syndromes (MDS). The accumulation of mitochondria harboring mtDNA mutations in patients with these disorders suggests a failure of normal mitochondrial quality-control systems. The mtDNA-mutator mice acquire somatic mtDNA mutations via a targeted defect in the proofreading function of the mtDNA polymerase, PolgA, and develop macrocytic anemia similar to that of patients with MDS. We observed an unexpected defect in clearance of dysfunctional mitochondria at specific stages during erythroid maturation in hematopoietic cells from aged mtDNA-mutator mice. Mechanistically, aberrant activation of mechanistic target of rapamycin signaling and phosphorylation of uncoordinated 51-like kinase (ULK) 1 in mtDNA-mutator mice resulted in proteasomemediated degradation of ULK1 and inhibition of autophagy in erythroid cells. To directly evaluate the consequence of inhibiting autophagy on mitochondrial function in erythroid cells harboring mtDNA mutations in vivo, we deleted Atg7 from erythroid progenitors of wildtype and mtDNA-mutator mice. Genetic disruption of autophagy did not cause anemia in wild-type mice but accelerated the decline in mitochondrial respiration and development of macrocytic anemia in mtDNA-mutator mice. These findings highlight a pathological feedback loop that explains how dysfunctional mitochondria can escape autophagy-mediated degradation and propagate in cells predisposed to somatic mtDNA mutations, leading to disease. (Blood. 2015;125(1):162-174)